Reorganization Of Exendin-4 Cloning, Expression And Purification

Exendin-4 (Incretin mimetic) is a polypeptide of 39 amino acids, separated from toxin ofsaliva glands of a kind of lizard, Gila monster lizard. It shares 53% amino acid homologywith glucagon-like peptide 1, GLP-1, which is essential to regulating blood sugar in type 2diabetes. Exendin-4 can induceβcell to release insulin by activating GLP-1 receptor. Sinceexendin-4 can hardly be degraded by Dipeptidyl PeptidaseⅣ(DDP-Ⅳ), it has muchlonger plasma half life and stronger and more stable hypoglycemic activity than GLP-1.Therefore, exendin-4 is a kind of drug with rosy perspective against type 2 diabetes.Currently, it is costly to synthesize polypeptides artificially. Therefore, a set of DNArecombinant techniques to express and purify recombinant exendin-4 in E coil systemwith high efficiency is desired to be established in our project. In this study, exendin-4gene with enterokinase recognition site closely at its N terminus and stop codon at the Cterminus was synthesized artificially, then it was constructed into vector pET-32a (+). Thusexpression vector pET-32a (+)-exendin-4, consisting of exendin-4, thioredox andhexahistidine, has been obtained. It was then used to transform Escherichia coli BL12(DE3) to produce expression strain, which was induced by IPTG. After ultrasonication ofthe thallus, lysate was purified through Ni+ affinity chromatograph, and fusion protein wasobtained. Then, recombinant exendin-4 was prepared through cleavage of exendin-4/Hisfusion protein with bovine enterokinase light chain (bovine-EKL) produced in Pichiapastoris system.As the results show, it is optimal to induce exendin-4/His fusion protein producing strainwith 0.1mM IPTG for 8hs at 30℃, producing target protein occupying above 16% of totalprotein in supernatant. And final yield of exendin-4/His fusion protein with more than 88%purity is 32mg/L. Yeast strain of multicopy bovine-EKL gene was obtained with G418YPD screening. And yeast supematant containing secretary bovine-EKL protein with highpurity was obtained, through 96hs' induction with 0.5% methanol at 30℃. Then it wasprecipitated with ammonium sulphate, and purified with anion exchange chromatographyso as to get bovine-EKL protein with nearly 100% purity. After ultrafiltration andconcentration, final product of 20mg/L yield (1.6μg/μl) was obtained. And 1067ng of thisfinal product has been shown to successfully cleave 50μg fusion protein. At last,exendin-4/His fusion protein was cleaved with bovine-EKL, releasing fragments of about4.2kDa, which is consistent with theoretical molecular weight of exendin-4.Our study shows that, fusion protein exendin-4/His can be highly expressed withpET-32a(+) system. Bovine enterokinase light chain expressed in Pichia pastoris yeastexpression system is active and can be used for cleaving fusion protein and preparingrecombinant exendin-4.In this study, an economical method with good performance for obtaining recombinantexendin-4 has been primitively established. And it is a good preparation for further study.